We have shown that endogenous eNOS-derived NO is critical for mediating vascular endothelial growth factor (VEGF)-induced placental endothelial cell angiogenesis and exogenous NO from donors also stimulates placental endothelial cell proliferation. However, the mechanisms by which NO directly regulate endothelial function post-biosynthesis is surprising very scant. S-nitrosylation (SNO) is a ubiquitous redox sensitive posttranslational modification to form nitrosothiols via adduction of a nitric oxide (NO) moiety o reactive cysteines. SNO is capable of modulating the entire proteome and has been recently recognized as a critical route through which NO signaling is transduced, severing to stabilize and diversify NO-related signals. In this RO3 grant, we seek seed funding to determine the changes in global protein SNO in the proteomes of primary endothelial cells treated with VEGF and NO donors for testing a hypothesis that a quantitative proteomics approach can be developed based on SILAC/MS and BST to identify all SNO-proteins in paired proteomes with simultaneous identification of specific SNO-site(s) in each SNO-protein. Aim 1 will develop a biotin-switch technique-based quantitative proteomics approach using stable isotope labeling of amino acids in cell culture (SILAC)/mass spectrometry and Aim 2 will use this method to differentiate the global changes of the nitroso-proteomes in VEGF- from NO donor- treated endothelial cells and to identify the specific SNO-sites (reactive cysteines) in all the SNO-proteins simultaneously. The proposed novel research will develop a novel comprehensive but simple unbiased quantitative proteomics method ideal for analyzing global SNO changes in any cells and even any biological samples. The beauty of this approach is that it is capable of digging out all the SNO-proteins in paired proteomes with simultaneous identification of the specific SNO-site(s) in all SNO-proteins identified. Unbiased quantitative comparison of the changes in the VEGF- vs. GSNO-responsive proteomes is also critically important because this will provide fundamental databases of the complete SNO-protein networks affected by endogenous NO upon VEGF stimulation and exogenous NO from donors, which is a significant next step for defining NO functions via cGMP-independent pathways in endothelial cells related to pregnancy. Moreover, the novel functional proteomics method developed will be able to use for analyzing protein SNO in any biological samples. Overall, this research will shed lights on the understanding of the important cellular processes via SNO in endothelial cells.